1. Field of the Invention
The present invention relates to compositions and methods for the use of such composition and solution for delivering substances into the cytosol and nucleus of living cells. More specifically, the compositions are peptides that transport nucleotides, oligonucleotides and polynucleotides into the cytoplasm and nuclei of cells.
2. Background and Related Art
In the course of biological research and certain medical procedures it is desirable to introduce a variety of substances into the cytosol of living eukaryotic cells. However, most hydrophilic substances, including many drugs, antisense and antigene oligos, RNAs, DNAs, peptides, proteins, carbohydrates, and combinations thereof, enter eukaryotic cells primarily via endocytosis and are subsequently sequestered or degraded in lysosomes, with little or no intact substance achieving the desired entry into the cytosol of the cells.
One of the diseases in which applicant is researching is coronary heart disease. Coronary heart disease is caused by the atherosclerotic narrowing of the coronary arteries affecting nearly 14 million persons in the United States. Approximately 480,000 deaths in 1995 were caused by the disease and it is the leading cause of death in the United States today. One of the established causes of atherosclerosis is elevated cholesterol levels and elevations of the major protein responsible for carrying cholesterol-apolipoprotein B (apoB). In treating atherosclerosis and other diseases, a difficult and yet unsolved problem is an effective way to deliver a drug to the cytosol and nuclear organelles in living organisms.
Double-stranded RNA interference (RNA-i) is a process wherein a ribonucleic acid is used to interfere with the expression of a certain gene. The RNAi is used to silence a particular gene by inhibiting the formation of a protein at the post-translation step of the process.
In order to silence a particular gene, the RNAi must enter the cell. There are several reagents and mechanisms by which this entry, called transfection, occurs. Examples of some known methods and reagents used in transfection include liposomes, small interfering RNAs (siRNAs), viral vectors (plasmids), RNA viruses, adeno viruses, lentil viral vectors, electrodeposition, and various custom-synthesized proprietary compounds.
Relevant background material is found in several scientific research articles. Among them are two articles published in the journal Nature; namely an “insight” review article entitled “RNA interference”, by Gregory J. Hannon in Volume 418, Jul. 11, 2002. This article discusses double-stranded RNA interference (RNA-i), cultivated as a means to manipulate gene expression experimentally and to probe gene function on a whole genome scale.
Biotech companies such as Ambion publish “TechNotes” to promote transfection of various siRNA expression vectors. Three brief articles appearing in the Ambion TechNotes newsletter include: A “How to” for New Users”, appearing in Volume 11, Number 5, 2004, “Delivering siRNA's to Difficult Cell Types, Volume 11, Number 3, November 2004, and siRNA Libraries Targeting Important Human Gene Classes, Volume 11, Number 5, 2004.
Several patents have been issued that include the use of viral vectors. They include U.S. Pat. No. 6,790,641 to Schauber et al, where the inventors provide a retroviral gene delivery system used to produce recombinant retroviral particles for transgene delivery. In U.S. Pat. No. 6,620,805 to Takle et al relates to porphyrins which protect the compound being delivered and delivers it to certain cell and tissue types. Porphryns are not categorized as peptides or proteins, but are a class of red-pigmented compounds with a cyclic tetrapyrollic structure in which the four pyrrole rings are joined through their alpha-carbon atoms by four methane bridges. The porphyrins form the active nucleus of chlorophylls and hemoglobin. Neither of these patents discusses the naturally occurring peptides that are the subject of this invention.
In U.S. Pat. No. 6,673,611 Thompson et al describe chemically-modified nuclei acid molecules; it is not about transport per se, as the novel nucleic acid molecules are not transport agents. In U.S. Pat. No. 6,617,438 to Beigelman et al the nucleic acids of interest have enzymatic activity and also are not transport agents.
In U.S. Pat. No. 6,828,149 Freier relates to antisense compounds and not transport agents or vectors. In U.S. Pat. No. 6,825,006 Baum et al again the modified nucleic acids are not transport agents.
In the present invention, Applicant is introducing a variety of nucleotides and nucleic acids into the cytosol of living eukaryotic cells. Applicant's invention deals with naturally occurring peptides that facilitate the transfer of nucleotides and nucleic acids into the nucleus and cytoplasm of cells.
The discovery of peptide, nucleotide and nucleic acid transporters that transport nucleic acids into the nucleus and cytosol of cells will permit the delivery of a variety of pharmaceuticals. Among the ailments thought to benefit from this invention is atherosclerosis, by regulating cholesterol metabolism. Cancer and other ailments will benefit from the delivery of nucleic acids as taught by the instant invention during chemotherapy. The peptides, due to their ability to transport ribonucleic acids into cells, can be employed in gene silencing by RNAi (ribonucleic acid interference) and antisense therapy.